C-terminal modification of polypeptides

ABSTRACT

The invention relates to a mutated trypsin comprising an amino acid substitution both at position K60 and D189, and at least one more amino acid substitution by histidine at position N143 or position E151. Such trypsin mutant has a preferred cleavage site comprising the amino acids Xaa 1 -Xaa 2 -His, wherein Xaa 1  is L, Y or F and Xaa 2  is R or K. The invention also relates to a man-made polypeptide comprising a target peptide and the above cleavage site as well as to a method of producing C-terminally modified target peptides by using this mutated trypsin.

RELATED APPLICATIONS

This application is a continuation of PCT/EP2005/008809 filed Aug. 12,2005 and claims priority to EP 04019237.9 filed Aug. 13, 2004.

BACKGROUND

The present invention relates to a mutated trypsin comprising an aminoacid substitution both at position K60 and D189, and at least one moreamino acid substitution by histidine at position N143 or position E151.Such trypsin mutant has a preferred cleavage site comprising the aminoacids Xaa₁-Xaa₂-His, wherein Xaa₁ is L, Y or F and Xaa₂ is R or K. Theinvention also relates to a man-made polypeptide comprising a targetpeptide and the above cleavage site as well as to a method of producingC-terminally modified target peptides by using this mutated trypsin.

The use of biologically active peptides, e.g. for pharmaceuticalpurposes has become more and more important during the past years.Several methods exist to produce such biologically active peptides, forexample, the chemical synthesis based on solid phase or solution phasepeptide synthesis techniques, or the cultivation of geneticallymanipulated microorganisms followed by the isolation and purification ofsuch produced recombinant proteins.

However, it remains difficult and costly to chemically synthesizepolypeptides of more than about 50 amino acids. It also represents asignificant task to modify a peptide obtained by chemical peptidesynthesis and/or a recombinantly obtained polypeptide at its C-terminalend. One of the most powerful methods to modify polypeptides is througha controlled protein ligation, whereby peptide analogs, unnatural aminoacids, stable isotopes, fluorophores, and other biochemically orbiophysically important molecules can be specifically incorporated intoa polypeptide. One of these methods is based on the introduction—mostlysynthetically—of a chemo-selective amino acid, mainly a cysteine whichthen is modified by a thio-selective reagent attacking the SH-side chainof this amino acid residue. A further alternative is the so-calledintein-based protein ligation system, which can generate a proteinthioester by proteolysis of a corresponding protein-intein fusionprotein (Blaschke, U.K., et al., Methods Enzymol. 328 (2000) 478-496).This method has been successfully applied to introduce unnaturalmodifications into proteins. However, difficulties remain, e.g., becausethe target protein must be expressed as a fusion protein together withan intein.

Recently a few more enzyme-based approaches for peptide ligation and/orC-terminal modification have been described. Breddam and co-workers(e.g., U.S. Pat. No. 5,985,627) describe the use of the serine proteasecarboxypeptidase Y (CPD-Y) in the C-terminal modification of peptideswith fluorescence or affinity labels. This modification is based on thespecific ability of CPD-Y to stepwise cleave amino acids off theC-terminal end of polypeptides. This method therefore may be consideredto be a specific tool for modification of the C-terminus of apolypeptide. CPD-Y cleaves off the C-terminal amino acid under formationof a peptide-acyl-enzyme-intermediate. This acyl-enzyme-intermediateupon nucleophilic attack is deacylated resulting in a transamidationreaction. The desired transamidation reaction may be accompanied by(un-wanted) side reactions like hydrolysis. It is also possible thatmore than one C-terminal amino acid is cleaved off, on the other handalso amino acids may be added by this method (Stennicke, H. R., et al.,Anal. Biochem. 248 (1997) 141-148 and Buchardt, O., et al., U.S. Pat.No. 5,580,751)

Abrahmson et al. (e.g. WO 94/18329) described the use of serine proteasevariants for ligation of peptides. Subtilisin variants are disclosedwhich have an improved peptide ligase activity. It is, however,necessary for effective peptide ligation to use an appropriate aminoterminus protecting group and an appropriate carboxy terminus activatinggroup, respectively, in order to efficiently ligate two peptidesubstrates.

Recently sortase-mediated protein ligation has been described as analternative method in protein engineering (Mao, H., et al., J. Am. Chem.Soc. 126 (2004) 2670-2671). Sortase, an enzyme isolated fromStaphylococcus aureus catalyses a transpeptidation reaction by cleavingbetween threonine and glycine in a recognition motif consisting of theamino acids LPXTG (SEQ ID NO: 22) and subsequently joining the carboxylgroup of threonine to an N-terminal glycine. In nature it catalyses thetranspeptidation of the threonine to an amino group of pentaglycine onthe cell wall peptidoglycan.

In the sortase recognition motif LPXTG (SEQ ID NO: 22), X may be theamino acids D, E, A, N, Q, or K. This enzyme has been used to ligatecarboxy terminal threonine residues of a peptide or a protein to anN-terminal glycine of a second peptide. As mentioned, sortase requires arecognition motif of five amino acids of which four amino acids (LPXT)will be present within the ligation product.

Therefore, whereas several methods exist for C-terminal modification ofpolypeptides there is a tremendous need for alternative or improvedmethods of C-terminal modification of polypeptides. The inventors of thepresent invention have found that it is possible to use special trypsinmutants in the C-terminal modification of polypeptides.

SUMMARY OF THE INVENTION

In one embodiment that present invention therefore relates to a mutatedtrypsin comprising an amino acid substitution both at position K60 andD189, and at least one amino acid substitution by histidine at positionN143 or position E151 according to the chymotrypsin nomenclature whichcorresponds to positions 43, 171, 123, and 131, respectively, of thesequence given in SEQ ID NO: 1.

The skilled artisan is familiar with the so-called chymotrypsinnomenclature as, e.g., described in Hartley, B. S., and Shotton, D. M.,The Enzymes, P. D. Boyer (ed.), Vol. 3, (1971), pp. 323-373 and willhave no problem in aligning the positions of a variant trypsin withpositions given according to the chymotrypsin nomenclature to thecorresponding ones of the trypsin sequence of SEQ ID No: 1.

Position 60 according to chymotrypsin nomenclature corresponds toposition 43 of the sequence of mature anionic rat trypsin II from Rattusnorvegicus as given in SEQ ID NO: 1.

Position 143 according to chymotrypsin nomenclature corresponds toposition 123 of the sequence of mature anionic rat trypsin II fromRattus norvegicus as given in SEQ ID NO: 1.

Position 151 according to chymotrypsin nomenclature corresponds toposition 131 of the sequence of mature anionic rat trypsin II fromRattus norvegicus as given in SEQ ID NO: 1.

Position 189 according to chymotrypsin nomenclature corresponds toposition 171 of the sequence of mature anionic rat trypsin II fromRattus norvegicus as given in SEQ ID NO: 1.

Since the skilled artisan is used to express positions referring to thechymotrypsin nomenclature, therefore, in the following the references tospecific sequence position, e.g., position K60 or simply position 60 areexclusively based on positions according to the chymotrypsinnomenclature.

The present invention also relates to the use of a man-made polypeptidecomprising a target peptide and a restriction site peptide comprisingthe cleavage site Xaa₁-Xaa₂-His, wherein Xaa₁ is L, Y or F, and Xaa₂ isR or K, wherein said restriction site peptide overlaps with the targetpeptide by the amino acid Xaa₁ at the C-terminal end of said targetpeptide as a substrate of a trypsin mutant as disclosed in the presentinvention.

Also provided is a method of producing a C-terminally transacylatedtarget peptide comprising the steps of: (a) providing a polypeptidecomprising a target peptide and a restriction site peptide comprisingthe cleavage site Xaa₁-Xaa₂-His, wherein Xaa₁ is L, Y or F, and Xaa₂ isR or K, wherein said restriction site peptide overlaps with the targetpeptide by the amino acid Xaa₁ at the C-terminal end of said targetpeptide, (b) bringing said peptide into contact with a trypsin mutantaccording to the present invention under conditions allowing forendoproteolytic cleavage after Xaa₁ and formation of an endoproteasetarget peptide-acyl-intermediate, (c) adding an appropriate nucleophile,and (d) upon nucleophilic attack and binding said nucleophile to theC-terminus of the target peptide releasing the mutated trypsin from theendoprotease target peptide-acyl-intermediate.

In a further embodiment the present invention relates to nucleotidesequences coding for the novel trypsin mutants, to vectors comprisingsuch mutants and to transformed host cells comprising such vectors.

The mutated trypsin according to the present invention is a trypsincomprising amino acid substitutions at both the positions K60 and D189and at least one amino acid substitution at position N143 or at positionE151. The substitution in position 143 and/or position 151 is byhistidine (His).

Preferably the mutated trypsin according to the present inventioncomprises either the amino acid E or the amino acid D in position 60,thus replacing the amino acid K normally present in position 60.

It is also preferred that the mutated trypsin according to the presentinvention comprises either the amino acid K, the amino acid H, or theamino acid R in position 189, thus replacing amino acid D normallypresent in that position. A very preferred substitution is with K atposition 189.

In a further preferred embodiment the mutated trypsin according to thepresent invention comprises mutations in positions 60, 143, 151 and 189.Preferred substitutions in this mutated enzyme are K60E or D, N143H,E151H and D189K or R.

The above described mutants of trypsin have very interesting andimportant properties. Such mutants appear to preferentially recognize abinding or cleavage site consisting of 3 amino acids Xaa₁-Xaa₂-His,wherein Xaa₁ is L, Y or F and Xaa₂ is R or K. This restriction site iscleaved by the above mutants after Xaa₁. This is a very importantfeature, because by using the novel trypsin mutants only one amino acid,i.e. the C-terminal Xaa₁, will remain within the modified targetpolypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Cloning vectors used for expression of mutant trypsinogen

On the left a schematic for the E. coli shuttle vector pST, comprising acoding region for trypsinogen is shown. The coding region can be easilyinserted into a pYT-vector, optimized for polypeptide expression inyeast. A schematic of a pYT-vector comprising a coding region fortrypsinogen is given on the right hand of this figure.

FIG. 2: Kinetics of transamidation

Time course of the transamidation of Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ IDNO: 2, triangles) with Arg-NH2 catalyzed by the trypsin variant Tn K60E,E151H, N143H, D189K resulting in Ala-Ala-Tyr-Arg-NH2 (SEQ ID NO: 23,circles) in the presence of a) 100 μM EDTA or b) 100 μM ZnCl2. Squaresrepresent AAY, which is formed as a side product of reaction due toproteolysis.

FIG. 3: Influence of Zn²⁺ on catalytic activity. Figure discloses SEQ IDNOS 3-7, respectively, in order of appearance.

The influence of the presence (grey bars) or absence (black bars) ofZn²⁺ ions on the rate of peptide turnover by trypsin variant Tn K60E,E151H, N143H, D189K is shown.

FIG. 4: Influence of the recognition sequence on the rate of reactioncatalyzed by the trypsin variant Tn K60E, E151H, N143H, D189K-catalyzed

Variant trypsin according to the present invention has been tested forcatalytic activity upon peptide substrates with different peptidesequences at or close to the cleavage site. The initial rate (v) ofpeptide consumption is given in nM/min

FIG. 5: Mass spectrum of Bz-Ala-Ala-Tyr-Arg-His-Lys (6-CF)—OH (SEQ IDNO: 24)

DETAILED DESCRIPTION OF THE INVENTION

To facilitate an understanding of the invention, a brief discussion ofthe terminology used in connection with the invention will be provided.The present disclosure uses the terminology of Schechter, J., andBerger, A., Biochem. Biophys. Res. Commun. 27 (1967) 157-162, todescribe the location of various amino acid residues on the peptidesubstrate and within the active site of a corresponding proteolyticenzyme.

According to the terminology proposed by Schechter, J. and Berger, A.,supra, the amino acid residues of the peptide substrate are designatedby the letter “P”. The amino acids of the substrate on the N-terminalside of the peptide bond to be cleaved (the “cleavage site”) aredesignated P_(n) . . . P₃, P₂, P₁ with P_(n) being the amino acidresidue furthest from the cleavage site. Amino acid residues of thepeptide substrate on the C-terminal side of cleavage site are designatedP₁′, P₂′, P₃′ . . . P_(n)′ with P_(n)' being the amino acid residuefurthest from the cleavage site. Hence, the bond which is to be cleaved(the “cleavage site”) is the P₁—P₁′ bond.

The generic formula for the amino acids of the substrate of anendopeptidase (like for example trypsin) is as follows:

P_(n)—P₃—P₂—P₁—P₁′—P₂′—P₃′—P_(n)′

The designation of the substrate binding sites of an endopeptidase isanalogous to the designation of amino acid residues of the peptidesubstrate. However, the binding sub sites of an endopeptidase aredesignated by the letter “S” and can include more than one amino acidresidue. The substrate binding sites for the amino acids on theN-terminal site of the cleavage site are labeled S_(n) . . . S₃, S₂, S₁.The substrate binding sub site for the amino acids on the carboxy sideof the cleavage site are designated S₁′, S₂′, S₃′, S_(n)′. Hence, in anendopeptidase, the S_(I)' sub site interacts with the P₁′ group of thepeptide substrate and the incoming nucleophile. A generic formula fordescribing substrate binding sites of an endopeptidase is:

S_(n)—S₃—S₂—S₁—S₁′S₂′—S₃′—S_(n)′

The S₁ binding site binds the side chain of the penultimate amino acid,P₁, of the peptide substrate, in case of a trypsin mutant according tothis invention the amino acid Xaa₁. The S₁′ binding site interacts withthe side chain of P₁′, in the present case with Xaa₂. Likewise, the S₂′binding site interacts with the side chain of the histidine residue inposition P₂′.

As the skilled artisan will appreciate the present invention may also becarried out with trypsin variants comprising an amino acid substitutionboth at position K60 and D 189, and at least one more amino acidsubstitution by histidine at position N143 or position E151.

The term “variant” refers to polypeptides having amino acid sequencesthat differ to some extent from a native polypeptide sequence.Ordinarily, a variant amino acid sequence will possess at least about80% homology with the corresponding parent trypsin sequence, andpreferably, it will be at least about 90%, more preferably at leastabout 95% homologous with such corresponding parent trypsin sequence.The amino acid sequence variants possess substitutions, deletions,and/or insertions at certain positions within the amino acid sequence ofthe native amino acid sequence. Preferably sequence homology will be atleast 96% or 97%.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “Align 2,” authored by Genentech,Inc., which was filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

Preferably, a variant of trypsin as disclosed in the present inventionin comparison to the corresponding wild-type sequence, comprises 20amino acid substitutions or less, more preferred 15 amino acidsubstitutions or less, also preferred 10 amino acid substitutions orless, and also preferred 6 amino acid substitutions or less.

The modified trypsin of the invention is capable of improvedtransacylation when compared to the corresponding native trypsin. Asused herein, “transacylation” is a reaction in which a peptide fragmentC-terminal to the trypsin cleavage site is exchanged for a nucleophile.Transacylation reactions include transthiolation, transesterificationand transamidation reactions. “Transamidation” occurs when an amide bondis formed between the nucleophile and the target peptide substrate. In atransamidation reaction, the nucleophile is not necessarily an aminoacid. “Transpeptidation” as an important subgroup of transamidationoccurs when the nucleophile is an amino acid, or amino acid derivative,such as an amino acid ester or amino acid amide.

A general transacylation reaction according to the present invention isshown below:

P_(n)—P₃—P₂—Xaa₁-Xaa₂-His-P₃′—P_(n)′+N→P_(n)—P₃—P₂—Xaa₁-N+Xaa₂-His-P₃′—P_(n)′

substrate+nucleophile mutated trypsin→modified substrate+cleaved offC-terminus

In the first step, the enzyme attacks the peptide bond between Xaa₁ andXaa₂, displacing the more C-terminal amino acids and forming a covalent(an acyl) bond between the P₁ residue (Xaa₁) of the target peptide andthe enzyme. This intermediate is referred to as a target peptide“peptide-acyl-enzyme intermediate” or briefly as acyl-enzymeintermediate. In the presence of an appropriate nucleophile, underproper conditions, the enzyme causes the nucleophile to add to thecleaved peptide substrate to produce a transacylated product. It isbelieved that the nucleophile attaches to the carboxyl group of theacyl-enzyme intermediate and displaces the enzyme from the acyl-enzymeintermediate. In this manner, the nucleophile becomes linked to thecarboxyl group of the peptide substrate.

Instead of undergoing a transacylation reaction, the acyl-enzymeintermediate might be deacylated by water to produce a hydrolysisproduct. The mutated trypsin of the invention is designed topreferentially produce the transacylation product over the hydrolysisproduct.

The present invention also relates to a man-made polypeptide comprisinga target peptide and a restriction site peptide comprising the cleavagesite Xaa₁-Xaa₂-His, wherein Xaa₁ is L, Y or F, and Xaa₂ is R or K,wherein said restriction site peptide overlaps with the target peptideby the amino acid Xaa₁ at the C-terminal end of said target peptide.With other words it relates to a man-made polypeptide comprising atarget peptide and a restriction site peptide comprising the cleavagesite Xaa₁-Xaa₂-His, wherein Xaa₁ is L, Y or F, and Xaa₂ is R or K,wherein said restriction site peptide and said target peptide share theamino acid Xaa₁ at the C-terminal end of said target peptide. Morepreferred Xaa₁ is Y or F, and especially preferred Xaa₁ is Y.

The term target peptide thus refers to the peptide or polypeptide of thesequence P_(n)'P₃—P₂—Xaa₁. The target peptide thus includes one aminoacid of the cleavage site for the mutated trypsin of this invention,i.e., the amino acid Xaa₁, being either L, Y or F. The term peptide doesinclude polypeptides.

The term man-made is used to indicate that the peptide sequence isartificial, e.g. it has been designed by a scientist or by a computer.The present invention does not relate to naturally occurringpolypeptides comprising the above-defined sequence motif Xaa₁-Xaa₂-His.It merely relates, to man-made, e.g. synthetically or recombinantlyproduced polypeptides which have been designed to comprise both a targetpolypeptide as well as a restriction site peptide to comprise thecleavage site Xaa₁-Xaa₂-His with Xaa₁ and Xaa₂ as defined above.According to our definition the target polypeptide has the amino acidXaa₁ as its C-terminal amino acid. The restriction site peptidecomprises at least the amino acids Xaa₂-His and optionally C-terminalthereto further amino acids. Thus the cleavage site consisting ofXaa₁-Xaa₂-His overlaps with the target polypeptide by one amino acid(Xaa₁) and by two amino acids (Xaa₂-His) with the restriction sitepeptide. As this skilled artisan will appreciate in principle anypolypeptide comprising Xaa₁-Xaa₂-His wherein Xaa₁ and Xaa₂ are asdefined above, may be used as a substrate for the trypsin mutantsaccording to the present invention, e.g. in an effort of peptideligation or in an effort of C-terminal peptide transacylation, e.g. formodification and/or labeling purposes.

Preferably a target polypeptide according to the present inventionconsists of 20 to 2000 amino acids. Also preferred is a target peptideconsisting of 30 to 1500 amino acids. More preferred such targetpolypeptide consists of 40-1000 amino acids.

Preferred target polypeptides are polypeptides used in diagnostic or intherapeutic applications.

Preferred target polypeptides for example comprise specific bindingagents, like antibodies and fragments thereof. Also preferred arespecific binding agents obtainable by phage display (see e.g., Allen, J.B., et al., TIBS 20 (1995) 511-516).

The term antibody refers to a polyclonal antibody, a monoclonalantibody, fragments of such antibodies, as well as to genetic constructscomprising the binding domain of an antibody. Any antibody fragmentretaining essentially the same binding properties as the parent antibodycan also be used.

Preferably the target polypeptide comprised in a recombinant polypeptideaccording to the present invention is a therapeutically activepolypeptide. Such therapeutically active polypeptide preferably isselected from the group consisting of a therapeutic antibody,erythropoietin and an interferon. Preferably the therapeutic protein iserythropoietin or an interferon.

It is obvious to the skilled artisan that only such polypeptides will beused as target polypeptides which do not comprise the sequence motifXaa₁-Xaa₂-His, with Xaa₁ and Xaa₂ as defined above, as part of theirsequence N-terminal to this desired cleavage site. The skilled artisanwill have no problems in excluding those polypeptides having a potentialcleavage site for a mutated trypsin of the present invention. In thealternative such potential internal cleavage site sequence may bemodified by routine mutation and/or cloning techniques to change and/orremove such un-desired internal cleavage site.

In a preferred embodiment the polypeptide according to the presentinvention which comprises at or close to its C-terminus the sequenceXaa₁-Xaa₂-His, wherein Xaa₁ and Xaa₂ are as defined above, is producedby recombinant methods. The skilled artisan will have no problem toengineer any desired target polypeptide which is accessible torecombinant production in a way to comprise at or close to theC-terminus the above defined restriction site of Xaa₁-Xaa₂-His.

The restriction site peptide according to the present invention at leastcomprises the amino acids Xaa₂ (R or K)-His with Xaa₂ at its N-terminus.It may contain additional amino acids C-terminal thereto whichfacilitate for example recombinant production or easy purification. In afurther preferred embodiment the recombinant polypeptide will compriseas part of the restriction site peptide a so-called His-tag at itsC-terminal end which allows for an easy purification by well establishedchromatographic methods, e.g., use of hexa-His and Ni-NTA-chromatography(Hochuli, E., et al., J. Chromatogr. 411 (1987) 177-184).

Preferably the above described trypsin mutants are used in a method forC-terminal acylation of a peptide substrate. As the skilled artisan willappreciate, such peptide substrate will comprise a cleavage site for amutated trypsin according to this invention and the method will comprisethe steps of providing an appropriate peptide substrate, bringing saidpeptide substrate into contact with a trypsin mutant according to thepresent invention under conditions allowing for endoproteolytic cleavageafter Xaa₁ and formation of an endoprotease targetpeptide-acyl-intermediate, adding an appropriate nucleophile, and uponnucleophilic attack and binding of said nucleophile to the C-terminus ofthe target peptide releasing the mutated trypsin from the endoproteasetarget peptide-acyl-intermediate.

Preferably the above-described mutant trypsins and the above-describedpolypeptides comprising Xaa₁-Xaa₂-His, with Xaa₁ and Xaa₂, as definedabove, are used in a method of C-terminal polypeptide modification bytransacylation, e.g. in a method for peptide ligation. In a preferredembodiment the present invention therefore relates to a method ofproducing a C-terminally transacylated target peptide comprising thesteps of: (a) providing a polypeptide comprising a target peptide and arestriction site peptide comprising the cleavage site Xaa₁-Xaa₂-His,wherein Xaa₁ is L, Y or F, and Xaa₂ is R or K, (b) bringing said peptideinto contact with a trypsin mutant according to the present inventionunder conditions allowing for endoproteolytic cleavage after Xaa₁ andformation of an endoprotease target peptide-acyl-intermediate, (c)adding an appropriate nucleophile and (d) upon nucleophilic attack andbinding of said nucleophile to the C-terminus of the target peptidereleasing the mutated trypsin from the endoprotease targetpeptide-acyl-intermediate.

As used herein, a nucleophile is a molecule that donates a pair ofelectrons to an electron acceptor, in this case the α-carboxyl carbon ofthe peptide-acyl-enzyme intermediate, to form a covalent bond.

Preferably the nucleophile is selected from the group consisting ofprimary amines, imines, secondary amines, thiol and hydroxyl. Suitablenucleophiles for example include amino acids; amino acid derivatives,such as amino acid esters and amino acid amides; amines, such asammonia, or benzyl amines.

The terms “transacylation” or “transacylated” are used to indicate thatthe C-terminal amino acid of the target peptide (Xaa₁) is bound viacovalent bond to the nucleophile. Where the nucleophile is a thiol thetransacylation is a thiolation, where the nucleophile comprises ahydroxylic group the transacylation is a esterification and where thenucleophile is an amine the transacylation results in a transamidation.Transamidation reactions are very important and represent a preferredembodiment according to the present invention.

As the skilled artisan will readily appreciate, appropriate nucleophilesmay furthermore comprise modifications which introduce desiredproperties to the C-terminal end of an appropriate target polypeptide.Preferably the present invention relates to a nucleophile comprising amodification that is selected from the group consisting of a peptide, apeptide amide, a label, a labeled amino acid amide, a labeled peptide, alabeled peptide amide, a non-natural amino acid, and polyethyleneglycol.

The term label is well-known to the skilled artisan and can be anydesired structure of interest. Preferably such label can be selectedfrom any known detectable groups, such as dyes, luminescent labelinggroups such as chemiluminescent groups e.g. acridinium esters ordioxetanes, or fluorescent dyes e.g. fluorescein, coumarin, rhodamine,oxazine, resorufin, cyanine and derivatives thereof. Other examples oflabeling groups are luminescent metal complexes such as ruthenium oreuropium complexes, enzymes as used for CEDIA (Cloned Enzyme DonorImmunoassay, e.g. EP-A-0 061 888), and radioisotopes.

Another preferred group of labels of interest for example comprises onepartner of a bioaffinity binding pair. While performing an assay thiskind of label interacts specifically and preferably non-covalent withthe other partner of the bioaffinity binding pair. Examples of suitablebinding partners of bioaffinity binding pairs are hapten orantigen/antibody, biotin or biotin analogues such as aminobiotin,iminobiotin or destheiobiotin/avidin or streptavidin, sugar/lectin,nucleic acid or nucleic acid analogue/complementary nucleic acid,receptor/ligand e.g. steroid hormone receptor/steroid hormone. Preferredlabels within this group are selected from hapten, antigen and hormone.Especially preferred labels are haptens like digoxin and biotin andanalogues thereof.

Another group of preferred modifications are non-natural amino acids andderivatives thereof. Most interesting are non-natural amino acidscontaining functional groups, which are orthogonal to the natural aminoacids, e.g. aldehyde functions, hydrazines, hydrazides, azides, andα-halogen-ketones.

In case the nucleophile comprises polyethylene glycol (PEG), this PEGpreferably has a molecular weight in the range of 2,000 Da to 50,000 Da.The PEG may be linear or branched. More preferred the PEG will be in themolecular weight range from 10,000 Da to 40,000 Da. Preferably thenucleophilic group of such nucleophile comprising PEG will be anarginine or a lysine having a free N-terminal α-amino group. Thisarginine or this lysine also may be the N-terminus of a peptide.Preferably such pegylated nucleophile is selected from the groupconsisting of Arg-His-PEG, Arg-His-Ala-PEG, Lys-His-PEG, Lys-His-Ala-PEGand Arg-His-Xaa-PEG, wherein Xaa may be any natural or non-naturaldi-amino carboxylic acid. The PEG-modified di-amino carboxylic acid maycomprise one or two PEG molecule(s) bound to one or to both of theseamino groups, respectively. The skilled artisan may select or designother appropriate PEG-modified nucleophiles, like a pegylated cysteineand others.

According to procedures known in the state of the art or according tothe procedures given in the examples section and armed with the teachingof the present invention, it is now possible to obtain polynucleotidesequences coding for the trypsin mutants of the invention. Preferablythe mutated trypsin according to the present invention is expressed asan inactive precursor (zymogen) which is enzymatically cleaved to resultin the active enzyme. In a further embodiment the present inventionrelates to a nucleotide sequence coding for the mutated trypsincomprising an amino acid substitution at position both at K60 and D189,and at least one more amino acid substitution by histidine at positionN143 or position E151, respectively.

The present invention further includes an expression vector comprising anucleic acid sequence according to the present invention operably linkedto a promoter sequence capable of directing its expression in a hostcell.

The present invention further includes an expression vector comprising anucleic acid sequence according to the present invention operably linkedto a promoter sequence capable of directing its expression in a hostcell. Preferred vectors are plasmids such as pST and pYT shown in FIG.1.

Expression vectors useful in the present invention typically contain anorigin of replication, a promoter located upstream in the DNA sequence,and are followed by the DNA sequence coding for a trypsin mutant,followed by transcription termination sequences and the remainingvector. The expression vectors may also include other DNA sequencesknown in the art, for example, stability leader sequences which providefor stability of the expression product, secretory leader sequenceswhich provide for secretion of the expression product, sequences whichallow expression of the structural gene to be modulated (e.g., by thepresence or absence of nutrients or other inducers in the growthmedium), marking sequences which are capable of providing phenotypicselection in transformed host cells, and the sequences which providesites for cleavage by restriction endonucleases.

The characteristics of the actual expression vector used must becompatible with the host cell, which is to be employed. For example,when cloning in an E. coli cell system, the expression vector shouldcontain promoters isolated from the genome of E. coli cells (e.g., lac,or trp). Suitable origins of replication in E. coli various hostsinclude, for example, a ColE1 plasmid replication origin. Suitablepromoters include, for example, lac and tip. It is also preferred thatthe expression vector includes a sequence coding for a selectablemarker. The selectable marker is preferably an antibiotic resistancegene. As selectable markers, ampicillin resistance, or canamycinresistance may be conveniently employed. All of these materials areknown in the art and are commercially available.

Suitable expression vectors containing the desired coding and controlsequences may be constructed using standard recombinant DNA techniquesknown in the art, many of which are described in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual (1989).

The present invention additionally concerns host cells containing anexpression vector which comprises a DNA sequence coding for the mutanttrypsin according to the present invention. Preferred are the host cellscontaining an expression vector comprising one or more regulatory DNAsequences capable of directing the replication and/or the expression of,and operatively linked to a DNA sequence coding for, all or a functionalpart of mutant trypsin. Suitable host cells include, for example, E.coli HB101 (ATCC 33694) available from Promega (2800 Woods Hollow Road,Madison, Wis., USA), XL1-Blue MRF available from Stratagene (11011 NorthTorrey Pine Road, La Jolla, Calif., USA) and the like.

Expression vectors may be introduced into host cells by various methodsknown in the art. For example, transformation of host cells withexpression vectors can be carried out by polyethylene glycol mediatedprotoplast transformation method (Sambrook et al. 1989). However, othermethods for introducing expression vectors into host cells, for example,electroporation, bolistic injection, or protoplast fusion, can also beemployed.

Once an expression vector containing trypsin mutant has been introducedinto an appropriate host cell, the host cell may be cultured underconditions permitting expression of the desired trypsin mutants. Hostcells containing an expression vector which contains a DNA sequencecoding for the trypsin mutant are, e.g., identified by one or more ofthe following general approaches: DNA hybridization, the presence orabsence of marker gene functions, assessment of the level oftranscription as measured by the production of trypsin mRNA transcriptsin the host cell, and detection of the gene product immunologically.

It should, of course, be understood that not all expression vectors andDNA regulatory sequences would function equally well to express the DNAsequences of the present invention. Neither will all host cells functionequally well with the same expression system. However, one of ordinaryskill in the art will make a selection among expression vectors, DNAregulatory sequences, and host cells using the guidance provided hereinwithout undue experimentation.

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Specific Embodiments EXAMPLE 1 General Procedure of Generating TrypsinVariants

-   1. Introduction of the desired mutations into trypsin or trypsinogen    using a suitable vector comprising the DNA encoding for trypsin or    trypsinogen, e.g., a pST vector (cf. FIG. 1).The E. coli vector pST    has been originally constructed from L. Hedstrom and represents a    yeast shuttle vector containing an ADH/GAPDH-promoter and α-factor    leader sequence fused to a sequence encoding for trypsinogen; see:    Hedstrom, L., et al., Science 255 (1992) 1249-1253.-   2. Transformation of the constructed vector e.g. in E. coli.-   3. Sub cloning of the modified trypsin-and trypsinogen sequence,    respectively, using suitable expression vectors, e.g. yeast vector    pYT (cf. FIG. 1). In the case the desired mutation was introduced in    this expression vector directly, this step is not needed.-   4. Expression of the modified trypsin or trypsinogen in E. coli or    yeast.-   5. Isolation of the modified trypsin or trypsinogen using suitable    separation methods, e.g. cation exchange chromatography.-   6. In the case trypsinogen has been expressed, the isolated zymogene    needs to be activated by limited proteolysis with enterokinase.-   7. Final purification of the activated trypsin applying suitable    purification methods, e.g. affinity chromatography or anion exchange    chromatography.-   8. Dialysis

In Table 1 the primary sequence of the trypsin variant Tn K60E, E151H,N143H, D189K (corresponding to mutated anionic rat trypsin II, withoutsignal sequence and pro-sequence) is given.

TABLE 1 Basic characteristics of the trypsinvariant K60E, N143H, E151H, D183K peptide sequence mass position (=SEQ ID NO: 1) 23828.5978 1-223 IVGGYTCQENSVPYQVSLNS GYHFCGGSLINDQWVVSAAHCYESRIQVRLGEHNINVLEG NEQFVNAAKIIKHPNFDRKT LNNDIMLIKLSSPVKLNARVATVALPSSCAPAGTQCLISG WGHTLSSGVNHPDLLQCLDA PLLPQADCEASYPGKITDNMVCVGFLEGGKKSCQGDSGGP VVCNGELQGIVSWGYGCALP DNPGVYTKVCNYVDWIQDTI AAN(Mutations are in bold)

Theoretical Peptide Mass:

[Theoretical pI: 5.41/Mw (average mass): 23843.00/Mw (monoisotopicmass): 23827.59]

EXAMPLE 2 Preparation of the Peptides by Means of Solid Phase PeptideSynthesis

Unless specified otherwise the peptides mentioned in this applicationwere synthesized by means of fluorenylmethyloxycarbonyl-(Fmoc)-solidphase peptide synthesis on a batch peptide synthesizer e.g. from AppliedBiosystems A433. In each case 4.0 equivalents of the amino acidderivative shown in Table 2 were used for this process.

TABLE 2 A Fmoc-Ala-OH C Fmoc-Cys(Trt)-OH D Fmoc-Asp(OtBu)-OH EFmoc-Glu(OtBu)-OH F Fmoc-Phe-OH G Fmoc-Gly-OH H Fmoc-His(Trt)-OH IFmoc-Ile-OH K Fmoc-Lys(Boc)-OH L Fmoc-Leu-OH M Fmoc-Met-OH NFmoc-Asn(Trt)-OH P Fmoc-Pro-OH Q Fmoc-Gln(Trt)-OH R Fmoc-Arg(Pbf)-OH SFmoc-Ser(tBu)-OH T Fmoc-Thr(tBu)-OH V Fmoc-Val-OH W Fmoc-Trp-OH YFmoc-Tyr(tBu)-OH

The amino acid derivatives were dissolved in N-methyl-2-pyrrolidinon.The peptide was synthesized on Wang resin (Wang, S.-S., J. Am. Chem.Soc. 95 (1973) 1328-1333) or on 2-chlortrityl chloride-resin (Barlos,K., et al., Tetrahedron Lett. 30 (1989) 3947-3950). The resin was loadedwith 0.5 to 1.0 mMol/g. The coupling reactions were carried out for 20minutes using 4 equivalents dicyclohexylcarbodiimide and 4 equivalentsN-hydroxybenzotriazole in dimethylformamide relative to the Fmoc-aminoacid derivative in dimethylformamide as the reaction medium. The Fmocgroup was cleaved after each step of the synthesis with 20% piperidinein dimethylformamide for 20 min. Terminal amino groups on the solidphase were optionally acetylated with acetic anhydride.

The introduction of a label e.g. a metal chelate label or a fluoresceinlabel or of a PEG at the C-terminus was carried out during the solidphase synthesis by the direct incorporation of for example a metalchelate or fluorescein coupled amino acid derivative (described in WO96/03409).

The peptide was released from the support and the acid-labile protectivegroups were cleaved with 20 ml trifluoroacetic acid, 0.5 ml ethanediol,1 ml thioanisole, 1.5 g phenol and 1 ml water within 40 min at roomtemperature. Depending on the amino acid derivatives that were used, itis also possible to use cocktails containing fewer radical traps. 300 mlcooled diisopropyl ether was subsequently added to the reaction solutionand was kept for 40 min at 0° C. in order to completely precipitate thepeptide. The precipitate was filtered, washed with diisopropyl ether anddissolved in a small amount of 50% acetic acid and lyophilized. Thecrude material obtained was purified by means of preparative HPLC onVydac RP C18 218TP152050 (column 50×250 mm, 300 Å; 15 μm) over anappropriate gradient (eluant A: water, 0.1% trifluoroacetic acid, eluantB: acetonitrile, 0.1% trifluoroacetic acid) within ca. 120 min. Theeluted material was identified by mass spectrometry.

Alternatively the label, e.g. PEG can also be introduced after cleavageof the peptide from the resin. For this it was advantageous to use achlortrityl chloride-resin. The protected peptide was cleaved off theresin with 1% trifluoroacetic acid in 10 ml dichloromethane for 20 minat room temperature. Then the C-terminus of the peptide was activated by2 equivalents dicyclohexylcarbodiimide, 2 equivalentsN-hydroxybenzotriazole and 2 equivalents triethylamine indimethylformamide as reaction medium and one equivalent of the aminoacid derivative of the labeling group or the effector group was added.The protective groups are removed by using 20 ml trifluoroacetic acid,0.5 ml ethanediol, 1 ml thioanisole, 1.5 g phenol and 1 ml water within40 min at room temperature. Depending on the amino acid derivatives thatwere used, it is also possible to use cocktails containing fewer radicaltraps. 300 ml cooled diisopropyl ether was subsequently added to thereaction solution and the reaction solution was kept for 40 min at 0° C.in order to completely precipitate the peptide. The HPLC purificationwas carried out as described above.

EXAMPLE 3 Transamidation of Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ ID NO: 2)with Arg-NH₂ Catalyzed by the Trypsin Variant Tn K60E, E151H, N143H,D189K

The peptide Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ ID NO: 2) was synthesizedby conventional solid-phase peptide synthesis using Fmoc-chemistry and apreloaded Wang-resin as described in Example 2. The respective aminoacid building blocks are commercially available and were purchased fromvarious suppliers. Arg-NH₂ was a commercial product from Bachem(Switzerland).

1 ml reaction volume containing 1 mM Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ IDNO: 2) and 5 mM Arg-NH₂ dissolved in 0.1 M Hepes buffer pH 8.0; 20 μMtrypsin variant Tn K60E, E151H, N143H, D189K; 100 μM ZnCl₂ or,alternatively 100 μM EDTA was stirred at 25° C. After defined timeintervals aliquots were withdrawn and reaction quenched by addition of1% trifluoroacetic acid in methanol/water (1:1, v/v) resulting in afinal pH of 2 of the withdrawn samples. The latter were analyzed byanalytical HPLC giving the time courses of the reactions as shown inFIG. 2. The identity of the final product of synthesis was verified bymass spectroscopy.

EXAMPLE 4 Influence of Zn²⁺ ions on the specificity of the trypsinvariant Tn K60E, E151H, N143H, D189K

The peptide substrates Bz-Ala-Ala-Tyr-Arg-His-Ala-Ala-Gly (SEQ ID NO:3), Bz-Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ ID NO: 4),Bz-Ala-Ala-Tyr-Arg-His-Asp-Ala-Gly (SEQ ID NO: 5),Bz-Ala-Ala-Tyr-Arg-Arg-Ala-Gly (SEQ ID NO: 6), andBz-Ala-Ala-Tyr-Asp-His-Ala-Gly (SEQ ID NO: 7)was synthesized byconventional solid-phase peptide synthesis using Fmoc-chemistry and apreloaded Wang-resin. The respective amino acid building blocks arecommercially available and were purchased from various suppliers.

1 ml reaction volume containing 1 mM of one of the following peptidesBz-Ala-Ala-Tyr-Arg-His-Ala-Ala-Gly (SEQ ID NO: 3),Bz-Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ ID NO: 4),Bz-Ala-Ala-Tyr-Arg-His-Asp-Ala-Gly (SEQ ID NO: 5),Bz-Ala-Ala-Tyr-Arg-Arg-Ala-Gly (SEQ ID NO: 6) orBz-Ala-Ala-Tyr-Asp-His-Ala-Gly (SEQ ID NO: 7) dissolved in 0.1 MPipes/Tris-buffer pH 8.0; 20 μM trypsin variant Tn K60E, E151H, N143H,D189K, 100 μM ZnCl₂ or, alternatively EDTA, was stirred at 30° C. Afterdefined time intervals, the reactions were terminated by addition of 1%trifluoroacetic acid in methanol/water (1:1, v/v). The quenched reactionmixtures were analyzed by analytical HPLC. The respective rates ofreactions are shown in FIG. 3. The identity of the final products wasverified by mass spectroscopy.

EXAMPLE 5 Influence of the Recognition Sequence on the Rate of ReactionCatalyzed by the Trypsin Variant Tn K60E, E151H, N143H, D189K

The N^(α)-benzoylated peptides of SEQ ID NOs: 3-19 have been synthesizedby conventional solid-phase peptide synthesis using Fmoc-chemistry and apreloaded Wang-resin. The respective amino acid building blocks arecommercially available and were purchased from various suppliers.

1 ml reaction volume containing 1 mM N^(α)-benzoylated peptide dissolvedin 0.1 M Pipes/Tris-buffer pH 8.0; 20 μM trypsin variant Tn K60E, E151H,N143H, D189K; 100 μM ZnCl₂ was stirred at 30° C. After defined timeintervals, the reactions were terminated by adding of 1% trifluoroaceticacid in methanol/water (1:1, v/v). The quenched reaction mixtures wereanalyzed by analytical HPLC. The respective rates of reactions are shownin FIG. 4. The identity of the final products was verified by massspectroscopy.

As can be easily seen from FIG. 4 the trypsin variant Tn (=K60E, E151H,N143H, and D189K) has a strong preference for a cleavage site comprisingL, F or Y in position P₁, R or K in position P₁′ and His in positionP₂′.

EXAMPLE 6 Transamidation of Bz-Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ ID NO:2) with Arg-His-Ala-Lys(6-CF)—OH (SEQ ID NO: 25) catalyzed by thetrypsin variant Tn K60E, E151H, N143H, D189K

Bz-Ala-Ala-Tyr-Arg-His-Ala-Gly (SEQ ID NO: 2) has been synthesized byconventional solid-phase peptide synthesis using Fmoc-chemistry and apreloaded Wang-resin. The respective amino acid building blocks arecommercially available and were purchased from various suppliers.Arg-His-Ala-Lys(6-CF)—OH (SEQ ID NO: 25) was synthesized by fragmentcondensation. The protected tripeptide Boc-Arg(Boc)₂-His(Trt)-Ala-OH wassynthesized on a chlorotrityl-resin using conventional solid phasepeptide synthesis. The peptide was cleaved from the resin with 2×40 mlof a cocktail containing methylene chloride/acetic acid/trifluoro aceticacid (v/v/v 8/1/1).

The crude material was purified by reversed phase HPLC. The synthesis ofthe other fragment, Fmoc-Lys(6-carboxy-fluorescein), was performed from0.6 mmol Fmoc-Lys*HCl, 0,655 mMol 6-carboxyfluorescein (purchased fromMolecular Probes) in 3 ml dioxin and 3 ml DMF. The Fmoc-group wascleaved off with piperidine and the crude material purified by reversedphase HPLC. Then the protected tripeptide was activated with 1equivalent of HBTU (Iris Biotech) and 3 equivalents ofdiisopropylethylamine in DMF. 1 equivalent of Lys(6-carboxy-fluorescein)was added and the mixture was stirred for 2 h at room temperature. Thenthe deprotection was done using a deprotection cocktail (18 mltrifluoroacetic acid, 0.5 ml water and 0.5 ml ethandithiol). The peptidewas precipitated with diisopropylether, purified by RP-HPLC, andobtained in good yield.

1 ml reaction volume containing 0.5 mM Bz-Ala-Ala-Tyr-Arg-His-Ala-Gly(SEQ ID NO: 20) and 2.5 mM Arg-His-Ala-Lys(6-CF)—OH (SEQ ID NO: 25)dissolved in 0.1 M Pipes/Tris-buffer pH 8.0; 20 μM trypsin variant TnK60E, E151H, N14311, D189K; 100 μM ZnCl₂ or, alternatively 100 μM EDTA,was stirred at 30° C. After defined time intervals respective aliquotswere withdrawn and quenched by addition of 1% trifluoroacetic acid inmethanol/water (1:1, v/v) resulting in a final pH of 2 of the withdrawnsamples. HPLC was used for analyzing the course of reaction andisolating the synthesis product. The latter was obtained in a yieldof >99% and has been further analyzed by mass spectroscopy as shown inFIG. 5.

EXAMPLE 7 Transamidation of AKTAAALHIL VKEEKLALDL LEQIICINIGADFGKLAKKHSIC PSGKRGGDLG EFRQGQMVPA FDKVVFSCPV LEPTGPLHTQ FGYHIIKVLY RH(SEQ ID NO: 21) with Arg-His-Gly-PEG catalyzed by the trypsin variant TnK60E, E151H, N143H, D189K

Arg-His-Gly-PEG is synthesized by fragment condensation ofBoc-Arg(Boc)₂-His(Trt)-Gly-OH and amino-PEG (20 kD) purchased formNektar/Shearwater. For synthesis of the protected tripeptide and theactivation of the fragment see example 6. Here 0.5 equivalents ofamino-PEG (20 kD) were used as nucleophile. After deprotection (cocktailsee example 6) all low molecular impurities were separated off usingRP-HPLC.

The polypeptide AKTAAALHIL VKEEKLALDL LEQIKNGADF GKLAKKHSIC PSGKRGGDLGEFRQGQMVPA FDKVVFSCPV LEPTGPLHTQ FGYHIIKVLY RH (SEQ ID NO: 21),containing the recognition sequence Tyr-Arg-His on its C-terminus, isproduced from native E. coli parvulin 10 by exchanging the originalC-terminal Asn moiety with an artificial His. After expression andpurification, the respective modified parvulin 10 (Asn92His) variant isdissolved in Pipes/Tris-buffer pH 8.0. Final concentrations of 20 μMtrypsin variant Tn K60E, E151H, N143H, D189K, 100 μM ZnCl₂, and anexcess of Arg-His-Gly-PEG are added into this reaction mixture. Afterstirring at 30° C. the reaction is terminated by addition of 1%trifluoroacetic acid in methanol/water (1:1, v/v). Analysis is done byHPLC, gel electrophoresis and/or mass spectroscopy. Isolation of thefinal product AKTAAALHIL VKEEKLALDL LEQIKNGADF GKLAKKHSIC PSGKRGGDLGEFRQGQMVPA FDKVVFSCPV LEPTGPLHTQ FGYHIIKVLY RH-Gly-PEG (SEQ ID NO: 26)is performed by conventional protein purification techniques, e.g. bychromatographic methods.

1. A mutated trypsin comprising an amino acid substitution at positionK60 and at position D189, and an amino acid substitution by histidine atposition N143 or position E151.
 2. The mutated trypsin of claim 1wherein K60 is substituted by E or D.
 3. The mutated trypsin of claim 1wherein D189 is substituted by K, H or R.
 4. (canceled)
 5. A method ofproducing a C-terminally transacylated target peptide comprising thesteps of: providing a polypeptide comprising a target peptide and arestriction site peptide comprising the cleavage site Xaa₁-Xaa₂-His,wherein Xaa₁ is L, Y or F, and Xaa₂ is R or K, wherein said restrictionsite peptide overlaps with the target peptide by the amino acid Xaa₁ atthe C-terminal end of said target peptide, bringing said peptide intocontact with a trypsin mutant according to claim 1 under conditionsallowing for endoproteolytic cleavage after Xaa₁, thereby forming anendoprotease target peptide peptide-acyl-intermediate, adding anappropriate nucleophile, and, upon nucleophilic attack and binding ofsaid nucleophile to the C-terminus of the target peptide, releasing themutated trypsin from the endoprotease target peptide-acyl-intermediate.6. The method of claim 5 wherein said nucleophile is selected from thegroup consisting of a primary amine group, an imine group, a secondaryamine group, a thiol group and a hydroxyl group.
 7. The method of claim5 wherein said nucleophile is selected from the group consisting of anamino acid amide, a peptide, a peptide amide, a label, a labeled aminoacid amide, a labeled peptide, a labeled peptide amide, andpolyethyleneglycol.
 8. The method of claim 7 wherein said nucleophile ispolyethyleneglycol.
 9. (canceled)